Accepted by the Acta Poloniae Pharmaceutica Drug Research
3.1.5 Discussion
Natural products in the form of crude or plant preparations have been used in the treatment and control of wide array of diseases. In many of such plants, some parts usually receive much attention compared to others. For instance A. melegueta, where most of the studies previously reported focused on seed alone, rendering the other parts to almost wastage. In this regard, we intend to explore the anti- oxidative and inhibition of key enzymes linked to T2D by various solvent extracts from fruit, leaf and stem of A. melegueta. This is of great importance as this will shade more light on the medicinal potentials of other parts of A. melegueta.
In previous studies, Adefegha and Oboh (2012a; 2011) have reported that, the seed aqueous extract of A. melegueta possessed higher polyphenolic and flavonoid contents, which was further supported in another study by Etoundi et al. (2010). Our data is partly in line with above-mentioned studies (Table 3.1). The higher polyphenolic and flavonoid contents of fruit EtOH extract compared to aqueous extract could be due variation in the method of extraction used. Additionally, in this present
study the polyphenolic and flavonoid contents of the aqueous extract were relatively similar with that of the seed aqueous extract reported earlier (Adefegha and Oboh, 2012a; 2011). Interestingly, leaf EtOH extract was also found to be rich in polyphenols and flavonoids contents. This is the first study that highlighted the contents of these phytochemicals from either the leaf or the stem parts. Furthermore, it is important to notice that, the amount and distribution of plants products such as polyphenols varies greatly from one species of plants to another or among the parts of the same plants. The production and accumulation of polyphenols are influenced by many factors, such as genetic and environmental factors (nature of the soil, high temperature and rain fall) in addition to growth or maturation stages for most of the parts of the plants (Mamphiswana et al. 2010; Pandey and Rizvi, 2009). Hence, the higher polyphenols and flavonoids contents of fruits compared to other parts could be attributed to one or combination of the above-mentioned factors, which were linked to most of the anti-oxidant ability of various plant-derived extracts.
In this study, a number of in vitro models were used to investigate the anti-oxidative potentials of various solvent extracts from different parts of A. melegueta. Among such model is the reduction of Fe3+ to Fe2+ by plant extracts or compounds is another reliable index and a good indicator of their electron-quenching abilities (Chung et al. 2002). This reaction is monitored by the formation of Perl’s Prussian blue color at 700 nm. In an attempt to further support the anti-oxidative nature of A. melegueta, Adefegha and Oboh, (2012b) have earlier reported higher reducing potential of A. melegueta aqueous seed extract, which correlate well with the results of our present study (Figure 3.1). Moreover, although no previous study is available on the A. melegueta leaf, our data have shown that leaf could be a good source of anti-oxidant in addition to seed and fruit that are popularly utilized. The higher ferric iron (Fe3+) reducing anti-oxidant power of various extracts from A. melegueta scavenging activity of EtOH extracts than other extracts from different parts of A. melegueta could be attributed to their rich polyphenols and flavonoids contents of these extracts, which has been further supported by the DPPH radical scavenging activity (Table 3.2).
The DPPH radical scavenging activity is a quick and reliable method for determining the anti- oxidative nature of plant-derived products. DPPH is a stable radical that usually delocalized by accepting electrons from the referenced anti-oxidant, thereby becoming a stable molecule (Lobo et al.
2010). The anti-oxidative effectiveness of various solvent extracts was assessed base on the calculated IC50 values. The lower the IC50 value, the higher the anti-oxidative potential of the extract. In some previous studies various extracts from the A. melegueta seed have shown good radical scavenging capacities. Kazeem et al. (2012) have reported an IC50 value of 0.110 ± 0.010 mg/mL for seed acetone extract, whereas 17.38 ± 2.00 mg/mL was earlier reported for seed aqueous extract (Adefegha and Oboh, 2012a). However, these are by far higher compared to that of fruit EtOH and aqueous extracts (EtOH: 0.04 ± 0.01mg/mL; aqueous: 0.04 ± 0.01 mg/mL) as well observed in our present study (Table 3.2). This has indicated higher electron scavenging ability of fruit than the seed part (Table 3.2).
Additionally, this is the lowest IC50 value reported for DPPH scavenging of any extracts derived from A. melegueta.
Hemoglobin glycation is a non-enzymatic reaction occurs when proteins are exposed to excess reducing sugars and contributes immensely to the formation of advanced glycation end products that lead to oxidative stress (Yamagishi et al. 2008; Rahbar and Figarola, 2003; Brownlee et al. 1984).
Kazeem et al. (2012) have earlier reported an IC50 value of 0.125 ± 0.02 mg/mL for acetone extract (0.25-1 mg/mL), which is obviously lower compared to that of fruit EtOH extract and other solvent extracts (Table 3.2). This could be due to the lower concentrations (30-240 µg/mL) used in our present study. The results were further supported by the α-amylase and α-glucosidase inhibitory actions of various extracts from A. melegueta (Table 3.2).
On the other hand, the control of postprandial hyperglycemia is crucial for the management of DM and prevention of its complications at early stage (Ali et al. 2006). This is achieved by delaying the absorption of glucose from the small intestine through the inhibition of carbohydrates metabolizing enzymes such as α-amylase and α-glucosidase, located at the intestinal tract. The α-amylase catalyzes the endo-hydrolysis of α-1, 4-glucosidic linkage releasing disaccharides and oligosaccharides which are further hydrolyzed at the small intestinal brush border by α-glucosidases to release glucose (Hua-Quang et al. 2012; Hanhineva et al. 2010). In a previous study, Adefegha and Oboh (2012a) have reported an IC50 value of 4.83 ± 0.56 mg/mL for α-amylase inhibition by seed aqueous extract. However, according to our results all the extracts derived from fruit part have demonstrated lower IC50 values compared to that of seed aqueous extract reported earlier, indicating better activity of the fruit than the seed part (Table 3.2). Similarly, the calculated IC50 value of the seed aqueous extract (2.14 ± 1.08 mg/mL) for α-glucosidase inhibition reported earlier was again far higher compared to that of fruit EtOH extract (0.06 ± 0.01 mg/mL) observed in our present study (Adefegha and Oboh, 2012a). Also observed in our data is that the IC50 value of the fruit aqueous extract was about 6-fold lower compared to that reported by Adefegha and Oboh (2012a) for seed aqueous extract. Furthermore, the relatively similar α- glucosidase inhibitory action of the leaf and fruit EtOH extracts has shown that leaf could be another potential source for α-glucosidase inhibitors (Table 3.2).
Based on the results of this study, fruit and leaf EtOH extracts that exhibited higher activities were subjected to GC-MS analysis to find the possible bioactive compounds that could be responsible for their activities. The study reported by Ilic et al. (2010) has indicated that, seed EtOH extract contains mainly the phenolic compounds such as gingerol, shogaol and paradol, which supports our present finding. It is also evident that eugenol (1), gingerol (6), capsaicin (7) 3-decanone, 1-(4-hydroxy-3- methoxyphenyl) (10) and ethyl homovallinate (11), detected in fruit and leaf EtOH extracts were of 4- hydroxy-3-methoxyphenyl derivatives. Therefore, it is propose that the presence of phenolics is regarded as the key feature that could be responsible for the higher anti-oxidative and anti-diabetic effects of the fruit and leaf EtOH extracts observed in this study in addition to the contribution of other compounds (Figure 3.7). Phenolics and other related compounds have the ability to act as anti-oxidants
due to their low reduction potential compared to highly reactive species such as hydroxyl (•OH), superoxide (O2•-), nitric oxide (NO•) radicals. Their ability to donate electron or proton from hydroxyl moieties result in stabilizing lipid peroxidation, neutralizing ROS and ultimately inhibit the initiation and propagation of chain reaction associated with oxidative damage (Brewer, 2011). Likewise, it has been proposed that phenolics interact with one or multiple sites of the α-glucosidase or α-amylase surface proteins and thus form a hydrophobic layer. This leads to aggregation and precipitation which changes the conformation of the enzyme structure and hence decrease in activity (Toda et al. 2001;
Spencer et al. 1988).